Pharmaceutical and/or cosmetic composition containing active-principle activators of aconitase

ABSTRACT

The present invention relates to a cosmetic or pharmaceutical, and in particular dermatological, composition comprising, in a physiologically suitable medium, active ingredients capable of activating aconitase. These active ingredients may be polypeptides or peptides, used alone or in combination with at least one other active ingredient. The invention also relates to the use of a cosmetic composition which protects mitochondria. The invention also relates to a cosmetic treatment process for protecting the skin and the appendages against external attacks and for combating skin ageing.

The present invention is in the cosmetic and pharmaceutical domain, andmore particularly in the domain of dermatology. The present inventionconcerns a cosmetic or pharmaceutical composition, and particularly adermatological one, comprising, in a physiologically adapted medium,active principles capable of activating aconitase. These activeprinciples may be polypeptides or peptides, used alone or in associationwith at least one other active principle. The invention is also relatedto the use of a composition capable of protecting mitochondria. Theinvention also concerns a cosmetic-treatment procedure intended toprotect the skin and the appendages from external aggressions and tocombat cutaneous aging. The said peptides, protectors of mitochondria,can also be used to prepare pharmaceutical compositions intended toprevent or combat pathologies linked to mitochondrial dysfunctions, forexample, certain neuromuscular or cardiac degenerations, type IIdiabetes, or even certain pathologies of aging.

The term “appendages” according to the invention encompasses theassemblage of keratinic appendices exhibited on the body surface, inparticular the hair, eyelashes, eyebrows, nails, and hair.

The mitochondria are organelles of eucaryote cells specialized in energyproduction. They are endowed with their own DNA (DNAmt). The energyproduced by the respiratory chain is stored in the form of ATP. Undercertain conditions of cellular stress, the electrons, released by therespiratory chain by oxidative phosphorylation produce free radicalsthat can damage the mitochondrial structures. These stress factors maybe intrinsic or of external origin. Among the latter, UV rays, toxins,radiation, and alimentary oxidants may be cited. Thus, a progressivedecline in mitochondrial functions with age can also be shown, probablylinked to the accumulation of mutations on DNAmt (K. Singh (2004), Ann.NY Acad. Sci., 1019). These DNAmt mutations can be induced by repeatedexposure to UV rays and will even be evidence of photo-aging (W.Berneburg et al. (2004), J. Invest. Dermatol. 122(5).

The mitochondria also play a central role in apoptosis. Mitochondrialdegradation may thus be a factor initiating the series of reactionsleading to apoptosis in numerous cell types (G. Kroemer et al. (1995),FASEB J. 9: 1277-87.

Natural repair mechanisms have been identified in the mitochondria.Aconitase (aconitase hydratase or iron regulatory protein; EC 4.2.1.3)has thus been discovered to be indispensible for the maintenance ofDNAmt integrity, independent of its catalytic activity. Aconitase wasfirst of all identified as a mitochondrial enzyme implicated in theKrebs cycle; it catalyzes the stereospecific conversion of citrate toisocitrate and thus participates in the energy production of the cell.It possesses an active site including an iron-sulfur aggregate (4Fe-4S)with a pseudocubic geometry. Recently, the team of Dr. Butow (X. J. Chenet al. (2005), Science 307) was able to show that aconitase plays a keyrole in preserving the integrity of mitochondrial DNA. It interacts withthe DNA within regulatory regions, suggesting a role in the packing ofthe mitochondrial genome in the manner of a protein in the HSP 70family. The transition from the form connecting the DNA to the activeform of the enzyme will be based on the assembling or disassembling ofthe iron-sulfur cluster.

Thanks to its double functionality, aconitase establishes a direct linkbetween the energy-generating activity of the mitochondrion and thepreservation of DNAmt. Aconitase could thus constitute a therapeutictarget for preventing or combating pathologies linked to mitochondrialdysfunctions, for example, type II diabetes, certain neuromuscularconditions, or certain pathologies of aging.

The search for compounds capable of stimulating or protecting themitochondria is an important concern of medical research and ofcosmetics. Concerning the skin, these new compounds could be useful inpreventing or combating the signs of cutaneous aging, as well as inprotecting or repairing the skin after burns, exposure to UV rays orradiation, or exposure to toxins or to pollutants.

In order to protect the mitochondria and to combat the associatedpathologies or the effects of cutaneous aging, solutions have beenproposed such as intake of substances implicated in energy metabolism,and more particularly, intermediaries or cofactors of the Krebs cyclesuch as fumarate, L-malate, acetyl CoA (WO 02064129), or even treatingthe skin with substances capable of reducing free radicals, such asvitamin C (US 2004/0086526) or L-ergothionine (WO 9836748). But, to theknowledge of the applicant, a composition containing active principlescapable of activating aconitase and its use, with the aim of protectingthe mitochondria and of combating the associated pathologies and/or theeffects of cutaneous aging, has never been described.

The present invention has a principal objective of providing a newactive principle capable of protecting the skin from externalaggressions and of combating cutaneous aging. The inventors have ineffect highlighted a therapeutic activity, and more particularly adermatological and cosmetic one, of peptides capable of activatingaconitase. It has been particularly brought out that these peptides,when they are applied on the skin, promote mitochondrial activity in asignificant way, demonstrated by an increased activity of the enzymeaconitase and an increase in the synthesis of ATP. These new activeprinciples, protectors of the mitochondria, thus open up new therapeuticand cosmetic perspectives.

Thus, the invention has as its first object a cosmetic or pharmaceuticalcomposition, characterized by the fact that it contains, as anactive-principle protector of the mitochondria, in a physiologicallyadapted medium, alone or in association with at least one other activeprinciple, a peptide capable of activating aconitase.

An “active-principle protector of mitochondria or capable of protectingmitochondria” is understood to be any substance capable of limiting thefunctional or structural alterations of cell or tissue mitochondriasubjected to a physico-chemical or environmental stress.

A “peptide capable of activating aconitase” is understood to be anypeptide or polypeptide capable of increasing aconitase, cytoplasmicand/or mitochondrial, either by activating the proteinic synthesis ofaconitase (by means of direct or indirect modulation of the geneexpression of the aconitase), or by increasing the enzymatic activity ofaconitase, or by other biological processes such as the stabilization ofthe aconitase protein or even the stabilization of the messenger RNAtranscripts.

The term “peptide” designates a chaining of two or several amino acidslinked to one another by peptide bonds or by modified peptide bonds, theterm “polypeptide” designating a peptide of greater size.

“Peptide” must be understood to be the natural or synthetic peptide ofthe invention as described above or at least one of its fragments, whichis either obtained by proteolysis or synthetically, or even any naturalor synthetic peptide whose sequence is wholly or partially composed ofthe sequence of the peptide according to the invention.

Preferentially, according to the present invention, theactive-principles activators of aconitase or their biologically activederivatives are peptides in which the number of amino acids is between 3and 14.

The expression “biologically active” is understood as “that possesses anin vivo or in vitro activity characteristic of the activity of theactive principle according to the invention”.

According to a particularly advantageous embodiment of the invention,the peptide possesses a sequence that answers to the general formula (I)

R₁-(AA)_(n)-X₁-X₂-X₃-X₄-X₅-X₆-(AA)_(p)-R₂

where

X₁ is serine,

X₂ is cysteine,

X₃ is threonine or isoleucine,

X₄ is glutamine or aspargine, or no amino acid,

X₅ is threonine, serine, or no amino acid,

X₆ is isoleucine, glutamine, or no amino acid.

-   AA represents any amino acid whatever, or one of its derivatives,    lacking the sequence of aconitase, and n and p are whole numbers    between 0 and 4.-   R₁ represents the primary amine function of the N-terminal amino    acid, free or substituted with a protector group which may be chosen    from an acetyl group, a benzoyl group, a tosyl group, or a    benzyloxycarbonyl group.-   R₂ represents the hydroxyl group of the carboxylic acid function of    the C-terminal amino acid, free or substituted with a protector    group which can be chosen from an alkyl chain of C₁ to C₂₀, or an    NH₂, NHY, or NYY group, with Y representing a alkyl chain of C₁ to    C₄.

According to one, most especially preferred embodiment of the invention,the biologically active peptide has the sequence:

(SEQ ID No. 1) Ser-Cys-Ile-Asn-Thr (SEQ ID No. 2)Ser-Cys-Ile-Asn-Thr-NH₂ (SEQ ID No. 3) Ser-Cys-Thr-Asn-Thr(SEQ ID No. 4) Ser-Cys-Thr-Asn-Thr-NH₂ (SEQ ID No. 5)Ser-Cys-Ile-Asn-Ser (SEQ ID No. 6) Ser-Cys-Ile-Gln (SEQ ID No. 7)Ser-Cys-Thr

According to one particularly interesting embodiment, the biologicallyactive peptide corresponds to the sequence SEQ ID No. 1.

According to another particularly interesting embodiment, thebiologically active peptide corresponds to the sequence SEQ ID No. 2.

The invention also concerns homologous forms of these sequences. Theterm “homologous” designates, according to the invention, any peptidesequence identical to at least 50%, or preferably to at least 80%, andeven more preferentially to at least 90%, of said peptide sequence,chosen from among the sequences SEQ ID No. 1 to SEQ ID No. 7. A “peptidesequence identical to at least X %” is understood to designate aidentity percentage between the residues of amino acids of the twosequences to be compared, obtained after the optimal alignment of thetwo sequences. The optimal alignment is obtained using algorithms forlocal homologies such as those used by the BLAST P or T BLAST N computersoftware available on the National Center for Biotechnology Information(NCBI) website.

The term “homologous” can also designate a peptide that differs from thesequence of a peptide with sequence SEQ ID No. 1 to SEQ ID No. 7 by thesubstitution of chemically equivalent amino acids, that is, by thesubstitution of one residue by another possessing the samecharacteristics. Thus, the classic substitutions are made between Ala,Val, Leu, and Ile; between Ser and Thr; between the acid residues of Aspand Glu; between Asn and Gln; and between the basic residues of Lys andArg or between the aromatic residues of Phe and Tyr.

In the invention, the term “amino acid” refers here to any natural orsynthetic organic acid having the formula:

—NHR—CR—C(O)—O—

where each —R is independently selected from a hydrogen or an alkylgroup having between 1 and 12 carbon atoms. Preferentially, at least one—R group of each amino acid is a hydrogen. The term “alkyl” isunderstood here to be a carbon chain which can be linear or branched,substituted (mono- or poly-) or unsubstituted; saturated, mono-saturated(a double or triple bond in the chain) or polyunsaturated (two orseveral double bonds, two or several triple bonds, one or several doublebonds and one or several triple bonds in the chain).

So as to improve resistance to degradation, it may be necessary toutilize a protected form of the peptide according to the invention. Theform of protection shall of course be a biologically compatible form andshall be compatible with a use in the domain of cosmetics or ofpharmaceuticals.

Numerous biologically compatible forms of protection may be envisioned.These are well-known to the professional, such as, for example, theacylation or acetylation of the amino-terminal end, or the amidation oresterification of the carboxy-terminal end. Thus, the invention concernsa composition as previously defined, characterized by the fact that thepeptide with sequence SEQ ID No. 1 to SEQ ID No. 7 is in a protectedform or not. A protection may be used which is based on a substitutionon the amino-terminal end by an acetyl group, a benzoyl group, a tosylgroup, or a benzyloxycarbonyl group. Preferably, a protection is usedwhich is based on the amidation of the hydroxyl function of thecarboxy-terminal end by an NYY group, with Y representing an alkyl chainof C₁ to C₄, or esterification by an alkyl group. It is also possible toprotect both the ends of the peptide.

The peptide derivatives also concern amino acids and peptides connectedto one another by a pseudo-peptide bond. A “pseudo-peptide bond” isunderstood to be all the types of bond likely to replace the “classic”peptide bonds.

In the domain of the amino acids, the geometry of the molecules is suchthat they can theoretically exhibit the form of different opticalisomers. Indeed, there exists one molecular conformation of the aminoacid (AA) such that it deflects the plane of light polarization to theright (dextrorotatory or D-aa conformation), and a molecularconformation of the amino acid (aa) such that it deflects the plane oflight polarization to the left (levorotatory or L-aa conformation). Thenature of the natural amino acids is such that it retains only thelevorotatory conformation. Consequently, a peptide of natural originwill be composed only of amino acids of the L-aa type. However, chemicalsynthesis in the laboratory enables amino acids to be prepared whichhave both possible conformations. Starting with this material as a base,it is thus possible, during peptide synthesis, to incorporate equallywell amino acids in the form of dextrorotatory or levorotatory opticalisomers. Thus, the amino acids composing the peptide according to theinvention may be in the L or D configuration; preferentially, the aminoacids are in the L form. The peptide according to the invention can thusbe in an L, D, or DL form.

The peptide with general formula (I) according to the invention can beobtained either by classical chemical synthesis (in solid phase orhomogeneous liquid phase) or by enzymatic synthesis (Kullman et al.(1980), J. Biol. Chem., 225, 8234), starting from the constituent aminoacids or from their derivatives.

The peptide according to the invention can also be obtained byfermentation of a strain of bacteria, modified or not by geneticengineering, or even by extraction of proteins of animal or vegetableorigin, preferentially of vegetable origin, followed by a controlledhydrolysis which releases peptide fragments which correspond in whole orin part to the peptides with general formula (I).

A great many proteins found in plants are likely to contain thesesequences within their structure. Controlled hydrolysis enables thesepeptide fragments to be released. It is possible, but not necessary toachieve the invention, either to extract the proteins concerned firstand then to hydrolyze them, or to perform the hydrolysis first on a rawextract and to subsequently purify the peptide fragments. It is alsopossible to use certain hydrolyzed extracts without purifying thepeptide fragments in them which correspond to peptides with the generalformula (I) according to the invention, but at the same time ensuringthe presence of the said fragments by appropriate analytical means.

Other procedures, simpler or more complex, may be envisioned by theprofessional familiar with the craft of synthesis, extraction, andpurification of proteins and peptides. Thus, the peptide according tothe invention can be of natural or synthetic origin. Preferentiallyaccording to the invention, the peptide is obtained by chemicalsynthesis.

According to the invention, the active principle can be a mixture ofpeptide derivatives and/or constituents of amino acid derivatives.

According to one advantageous embodiment of the invention, thepolypeptides or peptides with general formula (I) are solubilized inadvance in one or several cosmetically or dermatologically acceptablesolvents traditionally used by the professional, such as water,glycerol, ethanol, propylene glycol, butylene glycol, dipropyleneglycol, ethoxylated or propoxylated diglycols, cyclic polyols, vaseline,a vegetable oil, or any mixture of these solvents.

According to yet another advantageous embodiment of the invention, thepeptides possessing general formula (I) according to the invention aresolubilized in advance in a cosmetic or pharmaceutical vehicle like theliposomes or adsorbed onto powdered organic polymers or mineral supportslike the talcs and bentonites, and are more generally solubilized in, orfixed upon, any cosmetically or pharmaceutically acceptable vehicle.

It is of course understood that the peptide according to the inventioncan be used alone or in association with at least one other activeprinciple, in a cosmetic composition or for the preparation of apharmaceutical and/or dermatological composition.

The compositions according to the invention could be applied in anyappropriate way, particularly oral, parenteral, or externally topical,and their formulation will be adapted by the professional, in particularfor cosmetic or dermatological compositions. Advantageously, thecompositions according to the invention are intended for administrationby topical, cutaneous means. These compositions shall therefore containa cosmetically and/or dermatologically acceptable medium, that is,compatible with the skin and the appendages, and they cover all thecosmetic or dermatological forms. These compositions could, inparticular, be in the form of creams, oil-in-water or water-in-oilemulsions, or multiple emulsions, solutions, suspensions, gels, milks,lotions, sticks, or even powders, adapted to application onto the skin,the lips, and/or the appendages.

Of course, the professional will take care to choose possiblesupplementary compounds, active or not, and/or their quantity such thatthe advantageous properties of the mixture are not, or are notappreciably, altered by the addition envisaged.

These compositions include the excipients necessary for theirformulation, such as solvents, thickeners, diluents, surfactants,antioxidants, colorants, preservatives, or perfumes.

The composition usable according to the invention can, in particular,consist of a composition for hair care, and particularly a shampoo, aconditioner, a blow-dry lotion, a treatment lotion, a cream or a stylinggel, a restructuring lotion for the hair, a mask, etc. The cosmeticcomposition according to the invention can be used particularly intreatments implementing an application that is followed or not by arinse, or even in the form of shampoo.

It can also come in the form of a dye or a mascara to be applied withbrush or comb, in particular on the eyelashes, eyebrows, or hair.

Advantageously, the compositions usable according to the inventioncontain, in addition, other active principles promoting the action ofthe active principle according to the invention. For example, activeprinciples may be added which have an anti-radical or antioxidantaction, chosen from vitamin C, vitamin E, or coeznzyme Q10 or thepolyphenolic extracts of plants.

“Active anti-radical principles” are understood to be any compoundcapable of trapping free radicals. These active principles are capableof blocking the chain reactions of free radicals before the final stagesof degradation of the biological constituents of the skin, and becauseof this they have an antioxidant activity.

The composition according to the invention may also associate with thepeptide activators of aconitase active principles stimulating thesyntheses of dermal macromolecules (laminin, fibronectin, collagen), forexample the collagen peptide marketed under the name of Collaxyl® by theVincience Company.

The composition according to the invention may also associate with thepeptide activators of aconitase active principles stimulating energymetabolism, like the active principle marketed under the name of GP4G®by the Vincience Company.

From another angle, the composition according to the invention may be asun-related composition, that is, a composition that aids in protectionagainst solar radiation. Thus, there may be advantageously added to thecomposition according to the invention active agents which aid in solarprotection such as, for example, solar filters.

It is quite obvious that the invention is directed toward mammals ingeneral and more particularly toward human beings.

The effective amount of active principle corresponds to the quantitynecessary to obtain the result being sought, that is to say, to activateaconitase, protect the mitochondria, and thus protect the skin fromexternal aggressions and combat cutaneous aging.

According to an advantageous embodiment of the invention, the peptidewith general formula (I) is present in the compositions of the inventionin a concentration between approximately 0.0005 and 500 ppm (parts permillion), and preferentially in a concentration between approximately0.01 and 5 ppm, relative to the total weight of the final composition.

These compositions could come particularly in the form of an aqueous,hydroalcoholic, or oily solution, an oil-in-water or water-in-oilemulsion, or multiple emulsions. They can also come in the form ofcreams, suspensions, or even powders, adapted to application onto theskin, the mucous membranes, the lips, and/or the appendages. Thesecompositions can be more or less fluid and have the appearance of acream, a lotion, a milk, a butter, an ointment, a gel, a paste, or afoam. They can also come in solid form like a stick or be applied on theskin in the form of an aerosol. They can be used as a care productand/or as a makeup product for the skin.

These compositions include, in addition, any additive commonly used inthe application domain envisioned, as well as the adjuvants necessaryfor their formulation, such as solvents, thickeners, diluents,antioxidants, colorants, solar filters, self-bronzing principles,pigments, vehicles, preservatives, perfumes, odor absorbents, activecosmetic or pharmaceutical agents, essential oils, vitamins, essentialfatty acids, surfactants, film-forming polymers, etc.

In any case, the professional will take care that these adjuvants, aswell as their proportions, are chosen in such a way as not to harm theadvantageous properties sought in the composition according to theinvention. These adjuvants can, for instance, correspond to 0.01 to 20%of the total weight of the composition. When the composition of theinvention is an emulsion, the fatty phase may represent 5 to 80% byweight and preferably 5 to 50% by weight relative to the total weight ofthe composition. The emulsifiers and co-emulsifiers used in thecomposition will be chosen from among those traditionally used in thedomain considered. For example, they may be used in a proportion of 0.3to 30% by weight relative to the total weight of the composition.

By means of its specific activities, the peptide according to theinvention could be used advantageously in a cosmetic composition or forthe preparation of a pharmaceutical composition.

In particular, the active principle according to the invention could beused advantageously in a cosmetic composition intended to combat in apreventive and/or curative manner the manifestations of cutaneous agingand, more specifically, to combat and/or to prevent photo-induced aging(photo-aging). Cutaneous manifestations of aging are understood to beany alteration of the external appearance of the skin due to aging, suchas, for example, wrinkles and fine wrinkles, shriveled skin, flabbyskin, thin skin, lack of elasticity and/or tonus of the skin, dull skinwithout a glow or pigmentation spots on the skin, as well as anyinternal alteration of the skin that is not manifested systematically inan altered external appearance, such as, for example, any internaldegradation of the skin following an exposure to ultraviolet (UV) rays.The peptide according to the invention, or the composition containingit, will enable combating, in particular, the loss of elasticity andfirmness of the skin.

The peptide according to the invention may be used to protect the skinand the appendages against any type of external aggression. The use ofthe peptide, or of a composition containing it, will allow the skin andthe appendages to be protected and to better resist environmentalstresses.

The expression “external aggression” is understood to be aggressionswhich the environment can produce. By way of example, aggressions may becited such as pollution, UV rays, or even products of an irritatingnature such as surfactants, preservatives, and perfumes. Pollution isalso understood to be both “outdoor” pollution due, for example, toDiesel-fuel particles, ozone, or heavy metals, as well as “indoor”pollution, which may be due, in particular, to the emissions of thesolvents of paint, glue, or wallpaper (such as toluene, styrene, xylene,or benzaldehyde), or even cigarette smoke.

The peptide according to the invention may be advantageously used in acosmetic composition or for preparing a pharmaceutical composition, asan active photo-protective principle and, more particularly, as aso-called “secondary” active photo-protective principle. Primary activephoto-protective principles are, in effect, differentiated fromsecondary active photo-protective principles. Primary activephoto-protective principles are substances that exercise a physicalpower: they are capable of absorbing UV rays and releasing them in theform of heat in order to protect the skin. Secondary activephoto-protective principles are substances that generally have abiological effect; they are, for example, active principles capable oflimiting the damage caused to the DNA and to the membranes by UV rayspenetrating into the skin.

Thus, the invention also has, as an object, use in a cosmeticcomposition, or for preparing a pharmaceutical composition, of aneffective quantity of peptide as previously described, the peptide, orthe composition containing it, being intended to protect the skin fromexposure to UV rays or to ionizing radiation during radiotherapy.

The invention also has, as an object, use in a cosmetic composition, orfor preparing a pharmaceutical composition, of an effective quantity ofpeptide according to the invention, the peptide, or the compositioncontaining it, being intended to protect the mitochondria and toincrease the synthesis of intracellular ATP in the cells of the skin.

The invention too is related to use in a cosmetic composition, or forpreparing a pharmaceutical composition, of an effective quantity ofpeptide as previously described, the peptide, or the compositioncontaining it, being intended to protect the skin from damage caused byfree radicals.

The invention again consists of the use of an active principle accordingto the invention for preparing a pharmaceutical composition intended toprevent or combat mitochondrial dysfunctions responsible for certainneuromuscular or cardiac degenerations, type II diabetes, or evencertain pathologies of aging.

Finally, the invention again consists of a cosmetic-treatment procedureintended to protect the skin and the appendages from externalaggressions and to combat cutaneous aging, characterized by theapplication, onto the skin or the appendages to be treated, of acomposition containing an effective quantity of the active principleaccording to the invention.

Specific embodiments of this cosmetic-treatment procedure also resultfrom the preceding description. Other advantages and characteristics ofthe invention will be more apparent upon reading the examples given asillustrative and non-restrictive.

FIG. 1 represents the results of measuring the total enzymatic activityof aconitase in fibroblasts treated with SEQ ID No. 2 peptide andirradiated or not with UVB rays.

FIG. 2 represents the results of measuring specific enzymatic activityof mitochondrial aconitase in fibroblasts treated with SEQ ID No. 2peptide and irradiated or not with UVB rays.

EXAMPLE 1 Disclosure of the Stimulating Effect of the SEQ ID No. 1Peptide on the Synthesis of Intracellular ATP

The aim of this study was to determine the effect of the SEQ ID No. 1peptide on mitochondrial activity. It is the ATP produced by themitochondria that serves as a biochemical marker for this activity.

Protocol: This study was conducted using an ATP Bioluminescence AssayKit HS II (Roche Applied Science). Dermal fibroblasts were treated witha 1% solution of a 50-ppm solution containing the SEQ ID No. 1 peptide,representative of the peptide family according to the invention, for aperiod of 1 to 3 hours. At the end of the incubation time, the tubeswere emptied of their medium and rinsed with 2 ml of cold PBS beforeadding 250 μl of a lysing buffer provided in the kit. The cells of eachtube were then scraped up and collected in 14-ml tubes. Each tube wasrinsed with 2×500 μl of cold PBS, and the whole was collected again inthe respective tubes. Starting with these samples, a dilution to1/12000th was achieved in cold PBS before each reading. The ATPmeasurement was performed on these samples: 50 μl of this dilution wasplaced in a Luma cuvette and 50 μl of luminol was added. After 10seconds, the luminescence reading was begun. The values werestandardized relative to the quantity of proteins for each sample. Themeasurements were made using the Biocounter M2010A Luma®/3M equipment.

Results: The ATP measurements show that there was a clear increase inthe amount of intracellular ATP after one hour (+43%) and after 3 hoursin the cells treated with the SEQ ID No. 1 peptide, compared to theuntreated cells.

Conclusion: The SEQ ID No. 1 peptide very significantly stimulates themitochondrial activity of cutaneous cells as well as fibroblasts.

EXAMPLE 2 Disclosure of the Activator Effect of the SEQ ID No. 2 Peptideon the Enzymatic Activity of Aconitase

The aim of this study was to determine the effect of the SEQ ID No. 2peptide on the enzymatic activity of aconitase. For this, the totalenzymatic activity (cytoplasmic and mitochondrial) and the mitochondrialenzymatic activity of aconitase were measured.

Protocol: Normal human fibroblasts were treated with a 1% solution of a50-ppm SEQ ID No. 2 peptide representative of the peptide familyaccording to the invention, for 48 hr, and were then subjected to UVBradiation of 100 mJ/cm². After irradiation, the cells were treated underthe same conditions for another 24 hr. Negative controls were achievedunder the same conditions, but in the absence of the SEQ ID No. 2peptide.

To evaluate the enzymatic activity of aconitase, the fibroblasts werethen collected and centrifuged in PBS buffer. A portion of the cells wasthen processed to measure total enzymatic activity; another portion wasused to isolate the mitochondrial fraction according to the followingprotocol.

The cells were lysed using a Dounce-type homogenizer, and the homogenatewas centrifuged cold at 10,000 g for 10 minutes. The pellet, rich inmitochondria, was washed with TES buffer (10 mM Tris, 1 mM EGTA, 0.25 Msucrose) and resuspended in a 0.2-mM trisodium citrate buffer beforebeing used for the enzymatic measurement.

The measurement of the enzymatic activity of aconitase was made usingthe Oxis Bioxytech Aconitase-340 assay kit (Oxis International),according to the following principle: aconitase causes isomerization ofthe citrate to isocitrate. The isocitrate is then transformed intoα-ketoglutarate by the enzyme isocitrate dehydrogenase. This secondreaction is accompanied by the transformation of NADP+ to NADPH. It isthe formation of NADPH that is quantified spectrophotometrically at 340nm. Under the experimental conditions chosen, the rate of NADPHformation is proportional to the quantity of aconitase. The analysis ofthe kinetics of NADPH appearance enables the concentration of aconitasein the sample to be calculated (in mU/ml)

Results: The results derive from five independent experiments (n=5). Themeasurements of total enzymatic activity for aconitase (FIG. 1) show anincrease of 56.3% in the activity of cells treated with the SEQ ID No. 2peptide compared with untreated control cells. When the cells wereirradiated with UVB rays, on the other hand, a reduction of 33% wasobserved in this activity. The treatment before and after UVBirradiation with the peptide with SEQ ID No. 5 enabled the observedreduction to be limited in untreated control cells.

The measurements of the enzymatic activity of mitochondrial aconitase(FIG. 2) show comparable results.

The results are presented in FIGS. 1 and 2.

Conclusions: The SEQ ID No. 2 peptide, in a concentration of 0.05 ppm,strongly stimulates the enzymatic activity of the cytoplasmic andmitochondrial aconitase in cutaneous cells. The action of the SEQ ID No.2 peptide protects the cells and mitochondria from a UVB stressor.

EXAMPLE 3 Evaluation of the Protective Effect of the SEQ ID No. 2Peptide on Cutaneous Cells Subjected to Ultraviolet (UVB) Rays

The aim of this study was to determine the protective effect of the SEQID No. 2 peptide on dermal fibroblasts subjected to stress from UVBrays. For this, cellular viability tests were performed using the MTTtechnique.

Protocol: Human dermal fibroblasts were treated with a 1% solution of a50-ppm solution of SEQ ID No. 2 peptide for 24 hr, irradiated with UVBrays (25-100 mJ/cm²), and then cultivated for another 24 hr in thepresence of the same concentration of SEQ ID No. 2 peptide. Untreatedand non-irradiated controls, as well as untreated but irradiatedcontrols were achieved under the same conditions. At the end of theexperiment, the cells were incubated in a solution containing 0.1 mg/mlof MTT (3-[4,5-dimethylthiazolyl]-2,5-diphenyl-2H-tetrazolium bromide).This compound is absorbed by living cells and then metabolized by themitochondrial enzymes into a blue-violet compound, formazan, which ismeasured spectrophotometrically at 540 nm. The Absorbance (OD) is thendirectly proportional to the mitochondrial enzymatic activity as well asto the number of living cells.

Results:

Control without Control, Peptide + Control, Peptide + Control, Peptide +Conditions peptide Peptide UVB UVB UVB UVB UVB UVB SEQ ID — 0.5 0.5 —0.5 — 0.5 No. 2 peptide (ppm) UVB — — 25 25 50 50 100 100 irradiation(mJ/cm²) % 100 114.24 94.76 108.24 88.59 102.09 76.43 98.18 viability

Evaluation of cellular viability by the MTT technique showed, on the onehand, that the SEQ ID No. 2 peptide increases cellular viability,independent of any UVB radiation, and on the other hand, that UVBirradiation had a quantity-dependent cytotoxic effect on the humanfibroblasts, but that this cytotoxic effect was not exhibited in thepresence of 0.5 ppm of SEQ ID No. 2 peptide.

Conclusions: The SEQ ID No. 2 peptide, in a concentration of 0.5 ppm,increases cellular viability and, on the other hand, effectivelyprotects cutaneous cells against the cytotoxic effects of UVB rays.

EXAMPLE 4 Evaluation of the Protective Effect of the SEQ ID No. 2Peptide on Cutaneous Cells Subjected to Oxidative Stress

The aim of this study was to determine the protective effect of the SEQID No. 2 peptide with respect to dermal fibroblasts subjected to anoxidative stress caused by oxygenated water (H₂O₂) in a concentration of4 mM or 5 mM. For this, cellular viability tests were performed usingthe MTT technique.

Protocol: Human dermal fibroblasts were treated with a 1% solution of a50-ppm SEQ ID No. 2 peptide for 24 hours, subjected to an oxidativestress caused by 4 mM or 5 mM of H₂O₂ for 30 minutes, and thencultivated for another 24 hours in the presence of the sameconcentration of SEQ ID No. 2 peptide. Controls treated with neither thepeptide nor H₂O₂ and controls treated with the peptide alone wereachieved under the same conditions. At the end of the experiment, thecells were incubated in a solution containing 0.1 mg/ml of MTT(3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide)according to the protocol described in example 3

Results:

Control without Control, Peptide + Control, Conditions peptide PeptideH₂O₂ H₂O₂ H₂O₂ Peptide + SEQ ID —  0.5 —  0.5 — 0.5 No. 2 peptide (ppm)H₂O₂−, — — 4 mM 4 mM 5 mM 5 mM 30 min % 100 121.13 91.71 102.04 68.9294.40 viability

The evaluation of cellular viability by the MTT technique showed, on theone hand, that the SEQ ID No. 2 peptide increases cellular viability,independent of an oxidative stress, and on the other hand, that anoxidative stress caused by 4 mM or 5 mM of H₂O₂ for 30 min had aquantity-dependent cytotoxic effect on the human fibroblasts, but thatthis cytotoxic effect was not exhibited in the presence of 0.5 ppm ofSEQ ID No. 2 peptide.

Conclusions: The SEQ ID No. 2 peptide, in a concentration of 0.5 ppm,increases cellular viability and effectively protects cutaneous cellsagainst the cytotoxic effects of an oxidative stress.

EXAMPLE 5 Evaluation of the Cytoprotective Effect of the SEQ ID No. 2Peptide on Cutaneous Cells Subjected to Ultraviolet (UVB) Rays

A study of the cytoprotective effect of SEQ ID No. 2 peptide was made byevaluating the damage caused at the DNA level. This study was done usingthe comet (or single-cell gel electrophoresis) test, a quick andsensitive micro-electrophoretic technique that enables breaks in the DNAto be visualized and quantified in individual cells.

Protocol: Primary human fibroblasts were seeded in 100-diameter dishes.When the cells reached 70% coalescence, they were treated with SEQ IDNo. 2 peptide diluted to 1% (starting from a 50-ppm solution) for 24hours. The cells were then subjected to UVB irradiation at 100 mJ/cm²,then cultivated in the presence of the peptide for another 24 hours. Thedishes not treated with the peptide served as controls. A cellularsuspension of 100,000 cells/ml was then achieved, 500 μl were removed,inserted into an agarose solution, and run between slide and coverglass. The cells were lysed in the cold. The DNA was then denatured withan alkaline buffer and subjected to a brief electrophoresis (250 mA for30 minutes), and stained with propidium iodate. The slides were thenobserved under a fluorescence microscope. An altered cell sees its DNAstretch toward the anode in proportion to the number of breaks presentin the DNA, and it forms a “comet”. The highly degraded DNA is found inthe “tail” of the comet. An intact cell remains round, and its DNA isthen packed into the area of the comet “head”.

The evaluation of DNA breaks was done using image-analyzer software thatenables the percentage of DNA degradation to be determined, by means ofa quantitative evaluation of the “tail moment”, a parameter defined asthe product of the length of the comet by the percentage of DNA in itsdistal portion.

Results: The table below represents the evaluation of the “tail moments”measured in fibroblasts irradiated with UVB and treated or not with theSEQ ID No. 2 peptide.

Measurement of “tail % of “tail moment” Study conditions moment”relative to UV control Control, UVB 106,563 100 UVB + SEQ ID 80,221 75No. 2 peptide

The results obtained show that the cells subjected to UVB irradiation,in the absence of the SEQ ID No. 2 peptide (UVB control) exhibit thelargest “tail moment”, that is, the condition in which the DNA is mostdamaged. On the other hand, the cells treated with 0.5-ppm peptide withSEQ ID No. 2 have a “tail moment”, that is DNA damage, reduced by 25%.

Conclusion: The SEQ ID No. 2 peptide plays an important role inprotecting the DNA of cutaneous cells subjected to UVB irradiation.

EXAMPLE 6 Evaluation of the Action of the SEQ ID No. 2 Peptide on theUltra-Structure of Mitochondria

The aim of this study was to determine the action of the SEQ ID No. 2peptide on mitochondria, by observing the ultrastructures with theelectron microscope.

Protocol: Normal human keratinocytes were treated with a 1% solution ofa 50-ppm mother-solution of SEQ ID No. 2 peptide for 72 hours. Negativecontrols were achieved under the same conditions, but in the absence ofthe said peptide.

The cells were then fixed using Karnovsky's fixative (20 ml of 16%paraformaldehyde/8 ml of 50% glutaraldehyde/25 ml of 0.2 M sodiumphosphate buffer/25 ml of water) for 1 hr at ambient temperature, andthen left overnight at 4° C. The culture media were scraped into theKarnovsky's fixative and then the cells were centrifuged for 5 minutesat 5000 rpm. The supernatant was isolated and the pellet resuspended in1 ml of 0.1 M cacodylate. The samples were then kept at 4° C. untiltheir processing for electron microscopy observation.

Results: The organelles of the cells treated with SEQ ID No. 2 peptideexhibited ultrastructural characteristics suggesting an increase inmetabolic activity. In particular, the characteristics of themitochondrial population and the abundance of elements of the Golgiapparatus are in agreement with a stimulation of metabolic synthesis.

Conclusion: The SEQ ID No. 2 peptide has a stimulating action onmitochondrial activity.

EXAMPLE 7 Preparation of Compositions

1. Sunscreen Cream

International Nomenclature of Cosmetic Ingredients Trade name (INCI)names % w/w PHASE A Demineralized water Aqua (water) In sufficientquantity Pemulen ® TR-1 Acrylates/C10-30 alkyl 0.40 acrylatecross-polymer Glycerine Glycerin 3.00 Nipastat ® Sodium Sodiummethylparaben (and) 0.15 sodium ethylparaben (and) sodium butylparaben(and) sodium propylparaben (and) sodium isobutylparaben PHASE B Parsol ®MCX Ethylhexyl methoxycinnamate 7.50 Eusolex ® 4360 Benzophenone-3 3.00Parsol ® 1789 Butyl methoxydibenzoyl- 2.00 methane Myritol ® 318Caprylic/capric triglyceride 4.00 Emulgade ® SEV Hydrogenated palmglycerides 5.00 (and) ceteareth-20 (and) ceteareth-12 (and) cetearylalcohol Propylparaben Propylparaben 0.15 Nacol ® 16-98 Cetyl alcohol1.00 PHASE C TEA Triethanolamine 0.20 PHASE D SEQ ID No. 2 peptide 3 ppmPerfume Perfume (fragrance) In sufficient quantity Colorant Insufficient quantity

The constituents of phase A and of phase B are heated separately to atemperature between 70° C. and 75° C. Phase B is emulsified in phase Awhile stirring. Phase C is added, at 45° C., while increasing thestirring. Phase D is then added when the temperature was below 40° C.Cooling is continued to 25° C. with brisk stirring.

2. After-Sun Milk

Trade name INCI names % w/w PHASE A Montanov ™ L C14-22 alcohols (and)C12- 3.00 20 alkyl glucoside Waglinol 2559 Cetearyl isononanoate 4.00Tegosoft ® TN C12-15 alkyl benzoate 3.00 Apricot kernel oil Prunusarmeniaca (apricot) 2.00 kernel oil Avocado oil Persea gratissima(avocado) 1.00 oil Abil ® 350 Dimethicone 1.00 PHASE B Demineralizedwater Aqua (water) In sufficient quantity PHASE C Simulgel ™ EG Sodiumacrylate/acryloyl- 0.4 dimethyl taurate copolymer (and) isohexadecane(and) polysorbate 80 copolymer (and) polysorbate 80 PHASE D Phenonip ®Phenoxyethanol (and) 0.30 methylparaben (and) ethylparaben (and)butylparaben (and) propylparaben (and) isobutylparaben ethylparaben andpropylparaben and butylparaben Germall ® 115 Imidazolidinyl urea 0.20PHASE E SEQ ID No. 2 peptide 0.1 ppm

Prepare phase A while stirring. Incorporate the xanthan gum gradually,with dispersant stirring. Phases C and D will be incorporated once thegel has set. Phase E, prepared in advance to the point of perfect DHAdissolution, will then be added. Adjust the pH, if necessary, to 4-4.5.Color and perfume.

3. Anti-Aging Cream

Trade name INCI names % w/w PHASE A Montanov ™ 68 Cetearyl alcohol (and)6.00 cetearyl glucoside Squalane Squalane 3.00 Cetiol ® SB 45Butyrospermum parkii (Shea 2.00 butter) Waglinol 250 Cetearylethylhexanoate 3.00 Amerchol L-101 Mineral oil (and) lanolin 2.00alcohol Abil ® 350 Dimethicone 1.50 BHT Butylhydroxytoluene 0.01Coenzyme Q10 Ubiquinone 0.10 PHASE B Avocado oil Persea gratissima(avocado) 1.25 oil Phenonip ® Phenoxyethanol (and) 0.75 methylparaben(and)\ ethylparaben (and) butylparaben (and) propylparaben (and)isobutylparaben PHASE C Demineralized water Aqua (water) In sufficientquantity Butylene glycol Butylene glycol 2.00 Glucam ™ E10 Methylgluceth-10 1.00 Allantoin Allantoin 0.15 Carbopol ® Ultrez 10 Carbomer0.20 PHASE D TEA Triethanolamine 0.18 PHASE E SEQ ID No. 1 peptide 0.5ppm GP4G ® Water (and) Artemia extract 1.50 Collaxyl ® Water (and)butylene glycol 3.00 (and) hexapeptide-9 PHASE F Perfume Perfume(fragrance) In sufficient quantity Colorant In sufficient quantity

Prepare and melt phase A at 65-75° C. Heat phase C to 65-70° C. Phase Bis added to phase A just before emulsifying A in B. At about 45° C., thecarbomer is neutralized by the addition of phase D. Phase E is thenadded with light stirring, and cooling is continued to 25° C. Phase F isthen added, if desired.

4. Daytime Protection Cream

Trade name INCI names % w/w PHASE A Emulium delta ® Cetyl alcohol (and)glyceryl 4.00 stearate (and) PEG-75 stearate (and) ceteth-20 (and)steareth-20 Lanette O Cetearyl alcohol 1.50 DC 200 Fluid ®/100 csDimethicone 1.00 DUB 810C Coco caprylate/caprate 1.00 DPPG Propyleneglycol 3.00 dipelargonate DUB DPHCC Dipentaerythrityl 1.50hexacaprylate/hexacaprate Cegesoft ® PS 6 Vegetable oil 1.00 Vitamin ETocopherol 0.30 Phenonip ® Phenoxyethanol (and) 0.70 methylparaben (and)ethylparaben (and) butylparaben (and) propylparaben (and)isobutylparaben PHASE B Demineralized water Aqua In sufficient quantity,100 Glycerine Glycerin 2.00 Carbopol ® ETD 2020 Acrylates/C10-30 alkyl0.15 acrylate cross-polymer Keltrol CG-BT Xanthan gum 0.30 PHASE CSodium hydroxide (10% Sodium hydroxide 0.30 solution) PHASE DDemineralized water Aqua 5.00 Stay-C ® 50 Sodium ascorbyl phosphate 0.50PHASE E Butylene glycol Butylene glycol 2.00 Dekaben CP Chlorphenesin0.20 PHASE F GP4G ® Water (and) Artemia extract 1.00 SEQ ID No. 2peptide 5 ppm

Prepare phase A and heat to 75° C. while stirring. Prepare phase B,while dispersing the Carbopol®, and then the xanthan gum while stirring.Let rest. Heat to 75° C. At temperature, emulsify A in B withrotor-stator stirring. Neutralize with phase C while stirring rapidly.After cooling to 40° C., add phase D and then phase E. Cooling iscontinued with light stirring, and phase F is added.

1-17. (canceled)
 18. A cosmetic or pharmaceutical composition,comprising: a peptide in a physiologically acceptable medium, saidpeptide being an active-principle protector of mitochondria, alone or inassociation with at least one other active principle, wherein, saidpeptide is capable of activating aconitase or a biologically activederivative thereof, said peptide includes 3 to 14 amino acid residues,and said peptide has a sequence according to formula (I):R₁-(AA)_(n)-X₁-X₂-X₃-X₄-X₅-X₆-(AA)_(p)-R₂ in which X₁ is serine, X₂ iscysteine, X₃ is threonine or isoleucine, X₄ is glutamine, asparagine, orno amino acid, X₅ is threonine, serine, or no amino acid, X₆ isisoleucine, glutamine, or no amino acid, AA represents any amino acid,or derivative thereof, lacking sequence of aconitase, and n and p arewhole numbers between 0 and 4, R₁ represents the primary amine functionof the N-terminal amino acid, free or substituted with a protector groupselected from the group consisting of an acetyl group, a benzoyl group,a tosyl group, and a benzyloxycarbonyl group, R₂ represents the hydroxylgroup of the carboxylic acid function of the C-terminal amino acid, freeor substituted with a protector group selected from (i) an alkyl chainof C₁ to C₂₀ or (ii) an NH₂ group, an NHY group, or an NYY group, with Yrepresenting a alkyl chain of C₁ to C₄.
 19. The composition according toclaim 18, wherein said peptide has a sequence selected from the groupconsisting of: (SEQ ID No. 1) Ser-Cys-Ile-Asn-Thr; (SEQ ID No. 2)Ser-Cys-Ile-Asn-Thr-NH₂; (SEQ ID No. 3) Ser-Cys-Thr-Asn-Thr;(SEQ ID No. 4) Ser-Cys-Thr-Asn-Thr-NH₂; (SEQ ID No. 5)Ser-Cys-Ile-Asn-Ser; (SEQ ID No. 6) Ser-Cys-Ile-Gln; and (SEQ ID No. 7)Ser-Cys-Thr.


20. The composition according to claim 19, wherein said peptide has thesequence SEQ ID No.
 1. 21. The composition according to claim 19,wherein said peptide has the sequence SEQ ID No.
 2. 22. The compositionaccording to claim 18, wherein said peptide possesses at least onefunctional group protected by a protector group, and said protectorgroup is at least one of (i) an acylation or an acetylation of theamino-terminal end and (ii) an amidation or an esterification of thecarboxy-terminal end.
 23. The composition according to claim 18, whereinsaid peptide is present in the composition in a concentration betweenapproximately 0.0005 and 500 ppm.
 24. The composition according to claim23, wherein said peptide is present in a concentration between 0.01 and5 ppm.
 25. The composition according to claim 18, wherein said peptideis solubilized in advance in one or several cosmetically orpharmaceutically acceptable solvents selected from the group consistingof water, glycerol, ethanol, propylene glycol, butylene glycol,dipropylene glycol, ethoxylated or propoxylated diglycols, cyclicpolyols, vaseline, a vegetable oil, and mixture thereof.
 26. Thecomposition according to claim 18, wherein the physiological medium is acosmetically or dermatologically acceptable medium for topicalapplication.
 27. The composition according to claim 18, furthercomprising at least one other active principle promoting the action ofthe said peptide.
 28. The composition according to claim 27, wherein theother active principle is selected from the group consisting of activeantioxidant principles, active principles stimulating the synthesis ofthe extracellular matrix, active principles stimulating cellular energymetabolism, and combinations thereof.
 29. A method for protecting themitochondria and for increasing the synthesis of intracellular ATP inthe cells of the skin, comprising administering a cosmetic compositionaccording to claim 18 having an effective amount of active principle toa subject in need thereof.
 30. The method according to claim 29, whereinthe composition protects the skin and the appendages against externalaggression.
 31. The method according to claim 30, wherein the externalaggression is from UV rays.
 32. The method according to claim 29,wherein the composition prevents or treats the damage caused to the skinand to the appendages by free radicals.
 33. The method according toclaim 29, wherein the composition prevents or treats the cutaneous signsof aging and/or of photo-aging.
 34. A method of preparing apharmaceutical composition intended to prevent or to combatmitochondrial dysfunctions responsible for at least one of certainneuromuscular or cardiac degeneration, type II diabetes, and certainpathologies of aging, comprising to a physiologically acceptable medium,a peptide being an active-principle protector of mitochondria, alone orin association with at least one other active principle, wherein, saidpeptide is capable of activating aconitase or a biologically activederivative thereof, said peptide includes 3 to 14 amino acid residues,and said peptide has a sequence according to formula (I):R₁-(AA)_(n)-X₁—X₂—X₃—X₄—X₅—X₆-(AA)_(p)-R₂ in which X₁ is serine, X₂ iscysteine, X₃ is threonine or isoleucine, X₄ is glutamine, asparagine, orno amino acid, X₅ is threonine, serine, or no amino acid, X₆ isisoleucine, glutamine, or no amino acid, AA represents any amino acid,or derivative thereof, lacking sequence of aconitase, and n and p arewhole numbers between 0 and 4, R₁ represents the primary amine functionof the N-terminal amino acid, free or substituted with a protector groupselected from the group consisting of an acetyl group, a benzoyl group,a tosyl group, and a benzyloxycarbonyl group, R₂ represents the hydroxylgroup of the carboxylic acid function of the C-terminal amino acid, freeor substituted with a protector group selected from (i) an alkyl chainof C₁ to C₂₀ or (ii) an NH₂ group, an NHY group or an NYY group, with Yrepresenting a alkyl chain of C₁ to C₄.
 35. A method of protecting skinand appendages from external aggressions, comprising topically applyingto the skin or the skin appendages of a subject in need thereof, acosmetic composition according to claim 18 having an effective amount ofactive principle.